Epidermal growth factor signaling through transient receptor potential melastatin 7 cation channel regulates vascular smooth muscle cell function.

Zhi-Guo Zou,Rheure Alves-Lopes,Rhian M Touyz,George S Baillie,Vladimir Chubanov,Xing Gao,Thomas Gudermann,Jiayue Ling,Francisco J Rios,William Fuller,Augusto C Montezano,Livia L Camargo,Karla B Neves

doi:10.1042/cs20200827

Abstract

Objective: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function.Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR–TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice.Conclusions: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.

Highlights

Transient receptor potential (TRP) melastatin 7 (TRPM7) is a ubiquitously expressed bi-functional protein comprising a cation channel and a C-terminal α-kinase domain [1,2]
Using the carboxyfluorescein succinimidyl ester (CFSE) proliferation assay, we found that epidermal growth factor (EGF) induced proliferation of rat vascular smooth muscle cell (rVSMC) in an EGF receptor (EGFR)–TRPM7–ERK1/2-dependent manner, since EGF stimulated cell growth, effects that were inhibited by gefitinib, TRPM7 inhibitors and the ERK1/2 inhibitor, PD98059 (Figure 3A)
Further demonstrating a critical role for EGFR and TRPM7 in vascular homeostasis, we found that expression of Proliferating cell nuclear antigen (PCNA), a pro-prolifeartive marker and Notch3, a cell surface receptor expressed in vascular smooth muscle cell (VSMC) critically involved in vascular structure and development [44], was reduced in mouse vascular smooth muscle cell (mVSMC) and aortas from TRPM7+/ kinase mice (Supplementary Figure S4A–C)

Summary

Introduction

Transient receptor potential (TRP) melastatin 7 (TRPM7) is a ubiquitously expressed bi-functional protein comprising a cation channel and a C-terminal α-kinase domain [1,2]. TRPM7 is required to maintain cardiac automaticity in sinoatrial node (SAN) and regulates SAN fibrosis induced by angiotensin II (Ang II) through Smad signaling [9,10]. Using mice heterozygous for the truncation mutation in TRPM7 (TRPM7+/ kinase), we demonstrated that TRPM7 deficiency is associated with cardiovascular inflammation and fibrosis and increased susceptibility to Ang II-induced endothelial dysfunction and hypertension [11,12]. We found that TRPM7 plays an essential role in VSMC proliferation in a Mg2+-dependent fashion [14] and control of vasoactive agents such as Ang II and growth factors, including epidermal growth factor (EGF) [15]

Methods

Results

Conclusion